1H-Pyrazolo[3,4-b]pyridine-3-carbonitrile, 5-fluoro-1-[(2-fluorophenyl)methyl]-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-03-12 to 2019-09-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted March 23, 2006, corrected July 28, 2011

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD No 23, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures

Version / remarks:

February 08, 2019

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridine-3-carbonitrile

EC Number:

811-889-7

Cas Number:

1350653-26-7

Molecular formula:

C14 H8 F2 N4

IUPAC Name:

5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridine-3-carbonitrile

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Remarks:

LC-MS/MS

Details on sampling:

Duplicate samples from the freshly prepared test media (containing alga) of all test concentrations and from the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.
Additionally, samples were taken after 24 and 48 hours by taking aliquots of each replicate which were pooled.
The samples were diluted by a factor of two with acetonitrile.
Additional samples of the control and of the dilution solvent were taken at each sampling without any sample treatment.

Test solutions

Vehicle:

no

Details on test solutions:

Test Concentrations:
Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:3, 1:9, 1:27 and 1:81 of this filtrate were tested corresponding to following initially mean measured concentrations of the test item:
2.264, 0.752, 0.241, 0.0809 and 0.0258 mg test item/L, and a control. Control: In the control, test water was used without addition of the test item.
Dosage of Test Item:
The test item was not well soluble in test water. In this study, only the inhibitory effects of dissolved test item was tested and thus no concentrations above the solubility limit of the test item in test water were tested. Therefore, a supersaturated stock solution of nominal 100 mg test item/L was prepared by suspending 100 mg test item in 1000 mL test water for preparing the test concentrations. The stock suspension was shaken overhead for 24 hours at room temperature in the dark to dissolve as much test item as possible. Then, non-dissolved fractions of the test item were separated from the test medium by membrane filtration (0.45 µm cellulose acetate filter). The solution with dissolved test item was used as the test medium of the highest test concentration and to prepare the desired 1:3, 1:9, 1:27 and 1:81 dilutions.
The test media were prepared just before introduction of the algae (= start of the test).
Appearance of the Test Item in Test Medium: There were no remarkable observations.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG
Origin: The algae were supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of IBACON under standardised conditions according to the test guidelines.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

22°C

pH:

The pH was measured in all test item concentrations and the control at the start and the end of the test.
The pH of untreated test water was:8.0
The pH in the control was: 8.0 at test start and 8.9 at test end
The pH at different test item concentrations was: 8.0 at test start and 8.0 to 8.9 at test end.

Nominal and measured concentrations:

Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:3, 1:9, 1:27 and 1:81 of this filtrate were tested corresponding to following initially mean measured concentrations of the test item: 2.264, 0.752, 0.241, 0.0809 and 0.0258 mg test item/L, and a control.

Details on test conditions:

Type and Size: Erlenmeyer flasks of 50 mL volume with approximately 50 mL of test medium
Identification: Each test unit was uniquely identified with the serial number of the study, treatment and replicate number.
Test Conditions
Water Temperature: The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
Water temperature was: 22.1 to 22.4 °C
pH-Values:
The pH was measured in all test item concentrations and the control at the start and the end of the test.
The pH of untreated test water was: 8.0
The pH in the control was: 8.0 at test start and 8.9 at test end
The pH at different test item concentrations was:
8.0 at test start and 8.0 to 8.9 at test end
Light Regime: Continuous illumination
Light Intensity:
The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
Mean light intensity: 5257 lux (range: 4530 to 6010 lux) Recording: Test conditions were recorded with suitable instruments and documented in the raw data.

Reference substance (positive control):

yes

Remarks:

reference item potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.161 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.026 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.081 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.885 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.026 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.081 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Growth Inhibition:
The 72-hour EyC50 was calculated to be 0.161 mg test item/L and the ErC50 0.885 mg test item/L. The 72-hour EyC10 was calculated to be 0.040 mg test item/L and the ErC10 0.112 mg test item/L. The 72-hour NOEyC was determined to be 0.0258 mg test item/L and the associated 72-hour LOEyC of 0.0809 mg test item/L. The 72-hour NOErC was determined to be 0.0258 mg test item/L and the associated 72-hour LOErC is 0.0809 mg test item/L.
Signs of Phytotoxicity and Microscopic Observations:
The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a initially mean measured test concentration of 2.264 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.

Results with reference substance (positive control):

For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions. In the reference test with the reference item Potassium dichromate, performed in January 2019, the EC50 for yield was 0.445 mg test item/L, the EC50 for growth rate was 0.941 mg test item/L and the EC50 for biomass was 0.494 mg test item/L.

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hour ErC50 and the 72 hour EyC50 (see Definitions), the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis.
For the determination of the 72 hour LOEC and the 72 hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Bonferroni-Welch t-test (yield) and Williams t-test (growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Cell Density Increase: 180.3-fold increase within 72 hours; Coefficient of Variation of Sectional (Daily) Growth Rates: 11.0 %; Coefficient of Variation of Average Growth: 2.3 %

Conclusions:

The influence of Fluorpyridonitril on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 0.161 mg test item/L and the 72-hour ErC50 value was calculated to be 0.885 mg test item/L. The 72-hour NOEyC was determined to be 0.0258 mg test item/L and the associated 72-hour LOEyC was 0.0809 mg test item/L. The 72-hour NOErC was determined to be 0.0258 mg test item/L and the associated 72-hour LOErC was 0.0809 mg test item/L.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to initial mean measured values because the decrease of test substance could not quantifiably analysed in all samples.

Executive summary:

Title: Fluorpyridonitril: Toxicity to Pseudokirchneriella subcapitata in an Algal Growth Inhibition Test

Purpose:

The purpose of this test was to determine the inhibitory effect of the test item Fluorpyridonitril on the growth of the freshwater green algae Pseudokirchneriella subcapitata.

For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours hours, and thus over several algal generations. The test method of application and the test system are recommended by the test guidelines and Pseudokirchneriella subcapitata is one of the recommended test species.

The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

Guidelines/Recommendations:

OECD Guidelines for the Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted March 23, 2006, corrected July 28, 2011

OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals", 2nd Ed., February 08, 2019

SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

 

Materials and Methods

Test Item:

Fluorpyridonitril

Test Species:

Pseudokirchneriella subcapitata, Strain No. 61.81 SAG formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV).

Cultivated in the laboratories of ibacon; original source: "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissen-schaften, Universität Göttingen", 37073 Göttingen, Germany.

Test Design:

This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the controls. At test start approximately 50 mL of the test media were inoculated with nominal 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrical measurement.

Endpoints:

Yield and growth rate of the algae

Test Concentrations:

Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:3, 1:9, 1:27 and 1:81 of this filtrate were tested corresponding to following initially mean measured concentrations of the test item:

2.264, 0.752, 0.241, 0.0809 and 0.0258 mg test item/L, and a control.

Test Conditions:

Water temperature: 22.1 to 22.4 °C;

pH values in the control at test start: 8.0,

pH values in the control at test end: 8.9,

pH values in the test item treatments at test start: 8.0,

pH values in the test item treatments at test end: 8.0 to 8.9;

continuous illumination; mean light intensity: 5257 lux (4530 to 6010 lux).

Results

Biological Results:

Yield:

The 72-hour EC50 was calculated to be 0.161 mg test item/L. The 72-hour NOEC was determined to be 0.0258 mg test item/L and the associated 72-hour LOEC of 0.0809 mg test item/L.

Growth rate:

The 72-hour EC50 was calculated to be 0.885 mg test item/L. The 72-hour NOEC was determined to be 0.0258 mg test item/L and the associated 72-hour LOEC of 0.0809 mg test item/L.

Analytical Results:

The quantification of the test item Fluorpyridonitril in the test samples was performed using liquid chromatography with MS/MS detection.

The concentrations of the test item were determined in the duplicate test media samples from all test concentrations (test item stock solution and its dilutions) and the duplicate control samples from all sampling times. The initial mean measured values are

2.26 mg test item/L in the filtrate

0.752 mg test item/L in the 1:3 dilution of filtrate

0.241 mg test item/L in the 1:9 dilution of filtrate

0.0809 mg test item/L in the 1:27 dilution of filtrate

0.0258 mg test item/L in the 1:81 dilution of filtrate.

Conclusion:

The influence of Fluorpyridonitril on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 0.161 mg test item/L and the 72-hour ErC50 value was calculated to be 0.885 mg test item/L. The 72-hour NOEyC was determined to be 0.0258 mg test item/L and the associated 72-hour LOEyC was 0.0809 mg test item/L. The 72-hour NOErC was determined to be 0.0258 mg test item/L and the associated 72-hour LOErC was 0.0809 mg test item/L. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to initial mean measured values because the decrease of test substance could not quantifiably analysed in all samples.

This study is classified acceptable and satisfies the guideline requirements for growth inhibition algae studies.